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Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays
Published on: January 30, 2019
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Endotoxin recovery using limulus amebocyte lysate (LAL) assay
Jay S Bolden1, Rob E Warburton2, Robert Phelan3
1Global Quality Laboratories, Eli Lilly and Company, Indianapolis, IN, 46285, USA.
Summary
Low endotoxin recovery (LER) is a challenge for bacterial endotoxin testing (BET). Careful control of sample preparation procedures in spike/hold recovery studies is crucial for demonstrating the suitability of the Limulus Amebocyte Lysate (LAL) BET method for endotoxin detection.
Area of Science:
- Microbiology
- Biopharmaceutical Analysis
- Analytical Chemistry
Background:
- Low endotoxin recovery (LER) is a known phenomenon affecting bacterial endotoxin testing (BET).
- Regulatory agencies like the FDA require data on endotoxin recovery from spiked samples over time for Biologics License Applications (BLA).
- This is particularly relevant for drug substances (DS) and drug products (DP), including monoclonal antibody (Mab) products.
Purpose of the Study:
- To report on the experience and learnings from conducting spike/hold recovery studies for a Mab product.
- To investigate the impact of study design and execution on endotoxin recovery.
- To demonstrate the suitability of the LAL BET method (USP <85>) for endotoxin detection in biopharmaceutical products.
Main Methods:
- Conducting spike/hold recovery studies using a surrogate endotoxin (purified lipopolysaccharide - LPS) in a Mab product.
- Initial studies showed poor recovery; subsequent revisions focused on aligning study design with routine batch release USP <85> BET method parameters.
- Key parameters adjusted included mixing time and sampling scheme.
Main Results:
- Initial spike/hold studies with purified LPS exhibited rapid loss of endotoxin activity in the drug substance and significant batch-to-batch variation in the drug product.
- Revision of the study design to match the routine USP <85> BET method's mixing time and sampling scheme led to consistent LPS recovery over time.
- The revised studies demonstrated that LPS could be reliably recovered from the Mab product.
Conclusions:
- The initial challenges in endotoxin recovery were attributed to deviations in study design and execution from the validated USP <85> BET method.
- Careful control and standardization of sample preparation procedures, specifically mixing time and sampling, are critical for accurate spike/hold recovery studies.
- Properly controlled spike/hold studies are essential for demonstrating the suitability and reliability of the LAL BET method for endotoxin detection in biopharmaceutical development and manufacturing.
